ABSTRACT In this project we propose to exploit the recent successful isolation and reconstitution of the important ATP- dependent drug efflux transporter, ABCG2 (BCRP, Breast Cancer Resistance Protein), and combine it with a new transport assay system, the Fluorosome platform, to characterize the interaction of ABCG2 with drugs and drug candidates. The result will provide a useful, novel, highly specific and rapid in vitro transport assay for future general use. We shall employ this assay together with ATPase studies to characterize a wide variety of ABCG2 substrates and modulators. Additionally, by means of this unique construct we shall screen the 89 anticancer drugs in the NCI Oncology Drug Set Plate series for inhibition of ABCG2-mediated transport. The ABCG2 protein is a ubiquitous high capacity drug transporter with wide substrate specificity. The White Paper recently issued by the International Transporter Consortium formed to propose industry and FDA standards for studies of drug:transporter interactions lists ABCG2 as second in importance to P-glycoprotein (Pgp) among those drug transporters involved in clinical absorption and disposition of drugs. Drug effects are often modified by the highly variable oral uptake of drugs and by limited tissue distribution. Tumors frequently develop resistance against treatment with multiple chemotherapeutic agents. The consequence of this is the expectation that systemic chemotherapy of nearly one-half of the one million new cancer cases annually in the United States will fail due to the resistance of tumors to drugs. A major factor in this resistance is the prevalence of energy-driven efflux transporter proteins which actively pump drugs out of their target tissues. In addition, many side effects and incompatibilities accompanying multidrug use result from the interference of one drug with another's susceptibility to active efflux. The recent successful production, isolation, and reconstitution of the ABCG2 transporter combined with new technology employing the Fluorosome platform will provide an effective in vitro assay to determine the susceptibility of drug candidates to extrusion from their target tissue by the ABCG2 transporter and to determine if compounds interact with this transporter. These studies will employ a novel reagent Fluorosome-trans-abcg2 that is unambiguously specific for the ABCG2 transporter, applicable to a wide range of compounds, uses very small amounts of test material, and, with respect to instrumentation, requires only a standard injecting multiwell fluorescence plate reader. The assay will be amenable to moderate and high throughput screening.